The number of melanocytes in epidermis is very few as much as 1/10 of the number of keratinocytes. According to conventional separation methods, melanocytes are separated by: additionally treating an epidermal sheet, which is obtained by treating the skin tissue with dispase, with an enzyme such as trypsin to make single cells, and then providing a medium containing specific growth factors or chemokines to the cells to make only melanocytes survive. In respects that the melanocytes exist very few and there is no specific separation method except a medium, directly separating melanocytes from a living body is inefficient. For this reason, the price of commercial melanocytes is relatively expensive, compared with that of keratinocytes.
On the other hand, the melanocytes separated from a living body have limited homogeneity and proliferation capacity. Further, when using melanocytes in vitro shortly after directly separating the melanocytes from the epidermal sheet, it was reported that the cells have no response to stimulation such as UVB, or rather the expression of the genes related to melanin synthesis was reduced. In order to solve the above problems, measures, for example, treating UVB to co-culture of keratinocytes and melanocytes, or treating UVB to single-culture of keratinocytes followed by providing the culture to melanocytes, were needed.
It is needed to develop a method for separating melanocytes: which overcome the above shortcomings of the conventionally separated melanocytes, and thereby, having good homogeneity and proliferation capacity, and whose function can be studied without co-culturing them because their characteristics in vitro are maintained similar with the characteristics of when they are present in the body.
On the other hand, it is known that melanocytes are terminally differentiated from melanocyte stem cells, which are derived from neural crest during a developmental process, and melanoblasts as progenitor cells. The melanoblasts have better proliferation capacity and differentiation capacity than melanocytes, and have cell mobility (migration) as a characteristic of a developmental process.
Studies about development and differentiation from neural crest stem cell-melanoblast to melanocyte, and functions of the genes related to the above process have been done a lot in a mouse, but as a result of trial to separate melanoblasts by using some differentiation initial markers, yield was less than 5%. Furthermore, because the epidermal structure of the mouse is different from that of human, and there is no specific marker for dividing each differentiation steps, reports about methods for identifying the presence of melanoblasts in the human adult epidermis, or for directly separating melanoblasts from the skin tissue and culturing them are very rare. Some methods for inducing melanoblastic characters by changing a medium of the already separated melanocytes were reported, but it is hard to say that the cells are the melanoblasts existing in the human skin.